HORTSCIENCE 51(7):847–855. 2016.

Vermicompost Affects Soil Propertiesand Spinach Growth, Physiology, andNutritional ValueChenping Xu1 and Beiquan MouU.S. Department of Agriculture, Agricultural Research Service, U.S.Agricultural Research Station, 1636 East Alisal Street, Salinas, CA 93905

Additional index words. Spinacia oleracea, antioxidant, chlorophyll, drench, phytochemicals,soil amendment, worm castings

Abstract. The use of vermicompost to improve soil fertility and enhance crop yield hasgained considerable momentum due to its contribution to agroecological sustainability.Short-term (35 days after transplanting) effects of vermicompost, applied either as a soilamendment (5% and 10%, v/v) or a drench (40 mL of vermicompost extract at 0, 14, 21,and 28 days after transplanting), on soil properties and spinach plants (Spinacia oleraceaL.) were evaluated in a greenhouse. After harvesting, the amendments left high residuallevels of nutrients, organic matter and carbon, and increased soil cation exchange capacity(CEC) andwater-holding capacity (WHC). Drench treatment of unamended soil increasedsoil nutrients, CEC, andWHC. All vermicompost treatments, especially amendment at10% rate, increased leaf number, area, fresh and dry weight (FW and DW), shootFW and DW, root DW, and water use efficiency (WUE). Vermicompost increased leafchlorophyll content, and photochemical efficiency, yield, and electron transport rate(ETR) of mature leaves, as well as increased leaf succulence, and carotenoid, protein,and amino acid content. Vermicompost soil amendment reduced phenolics andflavonoids, leading to lower antioxidant capacity, whereas drench treatment onlydecreased betacyanin content. Vermicompost improved soil fertility, prompted leafproduction, delayed leaf senescence, and enhanced growth of spinach. It also favorablyinfluenced spinach quality by increasing leaf succulence and carotenoid, protein, andamino acids content, although it, as soil amendment, reduced flavonoid content leading tolow antioxidant capacity.

Soil organic matter plays a key role toachieve sustainability in agricultural pro-duction, because it possesses many desirableproperties such as high WHC, CEC, abilityto sequester contaminants, and beneficialeffects on the physical, chemical, and bi-ological characteristics of soil (Herrick,2000; Liu et al., 2006). In this context, theuse of organic soil amendments to improvesoil fertility and enhance crop yield hasgained considerable momentum for agro-ecological sustainability (D’Hose et al.,2014; Hargreaves et al., 2008).

Vermicomposting is a bio-oxidative pro-cess that uses earthworms and microorgan-isms for solid organic waste reclamation. The

microorganisms, both in the earthworm gutsand in the feedstock, are responsible forthe biochemical degradation of the organicmatter, whereas the earthworms are respon-sible for the fragmentation of the substrate,which increases the surface area exposed tothe microorganisms. The product, vermi-compost, is a finely divided mature peat-like material with high porosity, aeration,drainage, WHC, and microbial activity(Srivastava et al., 2011). It can be appliedas soil amendment to improve soil fertilityby increasing soil organic matter, CEC, andnutrient content, and improve soil structure(Arancon et al., 2006a; Srivastava et al.,2011). Many studies indicated that vermi-compost is preferable to compost to im-prove soil quality (Fornes et al., 2012;Tognetti et al., 2005).

There are many reports of positive effectsof vermicompost, as soil amendments orleachate, on many crops, including parsley(Petroselinum crispum Mill.) (Peyvast et al.,2008b), tomato (Solanum lycopersicum L.)(Arancon et al., 2003a, 2012), bell pepper(Capsicum anuum grossum L.) (Aranconet al., 2003a), lettuce (Lactuca sativa L.)(Arancon et al., 2012), mustard (BrassicaL.) (Srivastava et al., 2011), strawberry(Fragaria ananasa L.) (Arancon et al.,2003a, 2004), ryegrass (Lolium perenne L.)(Tognetti et al., 2005), sorghum (Sorghumbicolor L.) (Guti�errez-Miceli et al., 2008),

petunias (Petunias sp.) (Arancon et al., 2008),cow pea (Vigna unguiculataL.), banana (MusaacuminateL.), and cassava (Manihot esculentaL.) (Padmavathiamma et al., 2008). However,literature about the effects of vermicompost onspinach (Spinacia oleracea L.), an importantsalad vegetable with large quantities of bio-active compounds and nutrients, is veryscarce and focused on growth only (Peyvastet al., 2008a). Our objective was to assessthe short-term effects of vermicompost assoil amendments or leachate on soil proper-ties, and spinach growth, physiology, andnutritional value.

Materials and Methods

Plant materials and treatments. Two tri-als, eachwith four replications, were conductedfrom 30Mar. to 14May 2015 and 13Apr. to 28May 2015, in a greenhouse located in Salinas,CA (lat. 36�40#40$N, long. 121�39#20$W).The average temperature inside the green-house during the course of the trials rangedfrom 15 �C night to 34 �C day and relativehumidity ranged from 20% to 80%. Thegreenhouse was supplemented with light ofa 12-h photoperiod (Sun System 3; SunlightSupply, Vancouver, WA).

There were four treatments in this exper-iment: 1) Control: field soil (sandy loam)without amendments; 2) Drench: plantswere drenched with 40 mL of commercialliquid vermicompost extraction (WormPower, Avon, NY) at 0, 14, 21, and 28 dafter transplanting; 3) 5Ver: soil mixed with5% (v/v) of commercial granular vermicom-post (Worm Power, Avon, NY); 4) 10Ver:soil mixed with 10% (v/v) of granular vermi-compost. Plastic pots (diameter: 15 cm; depth:17 cm) with a single, bottom drain hole werefilled with 3 kg different mixture of soil andvermicompost amendments, and wateredjust to field capacity 2 weeks before trans-planting. Uniform-sized spinach seedlings(cv. Crocodile) were transplanted into pots10 d after sowing in rock wool cells (GrodanGroup, Roermond, Netherlands). Plants werethinned to one plant per pot 1 week aftertransplanting. Plants were irrigated twiceweekly and irrigation volumes were deter-mined by weighing each pot at field capac-ity and again just before irrigation. Theweight loss per pot was assumed to equaltotal evapotranspiration (ET), and its equiv-alent amount was applied for each pot.Therefore, the water applied was very closeto ET and the leached water and nutrientswere ineligible.

Soil and compost analysis. The untreatedfield soil and vermicompost samples werecollected before treatments were applied, andthe soil samples from different treatmentswere also collected using a soil sampler afterharvesting. One soil core (diameter: 2.6 cm;length: 15 cm) was collected from each potand four soil cores from each treatmentwere mixed together as one composite sam-ple for determination of macro- and micro-nutrients, pH, electrical conductivity (EC),organic matter and carbon, CEC, and WHC

Received for publication 15 Mar. 2016. Acceptedfor publication 8 May 2016.We thank Worm Power for providing vermicom-post products. The technical assistance of PhiDiep and Frances Wong, and critical review byJames McCreight and Renee Eriksen are greatlyappreciated.Mention of trade names or commercial products inthis publication is solely for the purpose of providingspecific information and does not imply recommen-dation or endorsement by the U.S. Department ofAgriculture.USDA is an equal opportunity provider andemployer.1Corresponding author. E-mail: chenping.xu@ars.usda.gov.

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by a commercial laboratory (Soil Control Lab-oratories, Watsonville, CA).

Growth and physiology measurements.Five weeks after transplanting in each trial,leaf maximum photochemical efficiency(Fv/Fm), photochemical yield [Y(II)], andETR were measured with a fluorometer

(MINI-PAM-II fluorometer; Heinz Walz,Effeltrich, Germany) on the first, second,and third pair of leaves from the bottom ofeach plant. Leaf Fv/Fm was measured afterleaves were adapted in darkness for 30 min.Then plants were harvested to measure leafnumber, area, FW and DW, shoot FW and

DW, and root DW. Sample DW was mea-sured after drying at 65 �C for 3 d. Leafarea was measured with a leaf area meter(CI-202 laser area meter; CID Bio-ScienceInc., Camas, WA). WUE was calculated asWUE = shoot FW/water used or ET.

Leaf discs were collected using a corkborer from the four largest leaves of eachplant to measure relative water content(RWC), specific leaf area (SLA), succu-lence, chlorophyll content, and nutritionalvalues. Specific leaf area was calculated asSLA = leaf area/DW (Evans, 1972). LeafRWC was calculated as RWC (%) = 100 ·[(FW – DW)/(TM – DW)], where TM isturgid mass after being soaked in water for4 h at 4 �C (Barr and Weatherley, 1962).Succulence was calculated as water con-tent per unit leaf area (Longstreth andNobel, 1979). Leaf pigments were extractedwith methanol and absorbance of theextraction was measured at 665, 652,and 470 nm (A665, A652, and A470) witha spectrophotometer (Spectronic Genesys;Spectronic Instruments, Rochester, NY).Chlorophyll a, b, and carotenoid contents(Ca, Cb, and Cx) were calculated using theformula described by Lichtenthaler (1987): Ca

(mg·L–1) = 16.72A665 – 9.16A652; Cb (mg·L–1) =34.09A652 – 15.28A665; Cx (mg·L–1) =(1000A470 – 1.63Ca – 104.96Cb)/221.

Phytochemical analyses. Leaf sampleswere soaked in liquid nitrogen immediatelyafter harvest and stored at –80 �C. Phyto-chemicals were extracted from �2 g ofsample material with 15 mL acidified meth-anol (1% HCl) using a homogenizer

Table 1. Physical and chemical properties of initial soil and granular vermicompost before treatment andsoil from each treatment after harvesting.

Properties

Initial After harvest

Soil Vermicompost Soil Drenchz 5Ver 10Ver

Total N (%) — 4.0 — — — —Available N (mg·kg–1) 48 — 6.0 7.0 6.0 10.0NH4-N (mg·kg–1) 4.6 17 4.7 4.7 4.4 6.6NO3-N (mg·kg–1) 43 8,000 <2 2.3 <2 3.2P (mg·kg–1) 39 7,300 32 40 51 61K (mg·kg–1) 79 34,000 67 80 140 240Ca (g·kg–1) 1.0 39 1.0 1.2 1.1 1.1Mg (mg·kg–1) 140 10,000 130 160 150 170SO4 (mg·kg–1) 32 6,200 16 25 24 48Cu (mg·kg–1) 0.53 1,000 0.56 0.72 2.0 2.8Zn (mg·kg–1) 2.6 250 2.9 3.5 3.4 3.8Fe (mg·kg–1) 32 3,400 25 39 33 53Mn (mg·kg–1) 13 180 12 14 10 9.6B (mg·kg–1) 0.27 53 0.30 0.38 0.42 0.44Na (mg·kg–1) 67 9,800 66 88 89 120Cl (mg·kg–1) 55 13,000 28 55 50 95pH 6.8 6.7 7.3 7.3 7.3 7.3ECy (dS·m–1) 1.9 26.0 0.58 0.91 0.92 1.4Organic matter (%) 2.3 72 2.4 2.5 2.9 3.4Organic carbon (%) 1.4 37 1.4 1.4 1.7 2.0Bulk density (g·mL–1) 1.22 0.26 1.17 1.22 1.18 1.14CEC (meq/100 g) 6.9 — 6.7 8.0 7.4 8.1WHC (g H2O/100 g soil) 7.00 — 6.85 7.62 8.16 7.74C:N ratio — 9.3 — — — —zDrench: 40 mL of vermicompost extract at 0, 14, 21, and 28 d after transplanting; 5Ver or 10Ver: soilamended with 5% or 10% (v/v) vermicompost.yEC = electrical conductivity; CEC = cation exchange capacity; WHC = water-holding capacity.

Fig. 1. Effect of vermicompost on spinach leaf number (A), area (B), fresh (C) and dry (D) weight 35 d after transplanting. The values are means of eight replicates±SE. Different letters on top of bars indicate significant difference atP# 0.05 according to Student’s t test. Drench: 40mL of vermicompost extract at 0, 14, 21,and 28 d after transplanting; 5Ver or 10Ver: soil amended with 5% or 10% (v/v) vermicompost.

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(Polytron; Kinematica AG, Schweiz, Swit-zerland), then incubated in darkness at –20 �Covernight. After centrifuging at 9070 gn for15 min, the supernatant was collected for theanalysis of nutrition values. Its A535 wasmeasured for total betacyanin content. Re-sults were calculated using a molar extinctioncoefficient of 65,000 (Schwartz and von Elbe,1980). The antioxidant capacity was mea-sured by the method of ferric-reducing abilityof plasma (Benzie and Strain, 1996). 10 mM

2,4,6-tris-2,4,6-tripyridyl-2-triazine (TPTZ)and 20 mM ferric chloride was diluted in300 mM sodium acetate buffer (pH 3.6) ata ratio of 1:1:10. Extracts (25 mL) wereadded to 2 mL TPTZ solution, and A593 wasdetermined after 4.5 min reaction. Trolox(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxyl acid) equivalent (TE) standardcurve was prepared.

For total phenolics content, 0.1 mL ex-tract was added to a mixture of 0.15 mL H2Oand 0.75 mL of 1:10 diluted Folin–Ciocalteureagent (Sigma-Aldrich, St. Louis, MO).After 6 min, 0.60 mL of 7.5% (w/v) Na2CO3

was added and vortexed, then the mixturewas incubated at 45 �C in a water bath for10 min. Samples were allowed to cool to roomtemperature before reading A765 (Slinkardand Singleton, 1997). A standard curve wasprepared from a freshly made gallic acidequivalent (GAE) solution. For total flavo-noid content, 0.20mL extract was mixed with

Fig. 2. Effect of vermicompost on spinach shoot fresh weight (A), water use efficiency (WUE; B), dry weight (C), and fresh:dry ratio (D) 35 d after transplanting.The values are means of eight replicates ±SE. Different letters on top of bars indicate significant difference at P# 0.05 according to Student’s t test. Drench:40 mL of vermicompost extract at 0, 14, 21, and 28 d after transplanting; 5Ver or 10Ver: soil amended with 5% or 10% (v/v) vermicompost.

Fig. 3. Effect of vermicompost on spinach root dry weight (A) and shoot:root ratio (B) 35 d aftertransplanting. The values are means of eight replicates ±SE. Different letters on top of bars indicatesignificant difference at P # 0.05 according to Student’s t test. Drench: 40 mL of vermicompostextract at 0, 14, 21, and 28 d after transplanting; 5Ver or 10Ver: soil amended with 5% or 10% (v/v)vermicompost.

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0.85 mL distilled water and 50 mL of 5%NaNO2. After 6 min, 100 mL of 10%AlCl3·6H2O was added, and after another5 min, 0.35 mL of 1 M NaOH and 0.20 mLdistilled water were added, then A510 wasmeasured immediately (Dewanto et al.,2002). A (+)-catechin hydrate equivalents(CHE) standard curve was prepared froma freshly made solution.

Protein and amino acid contents. Leafsamples (about 2 g) were homogenized in15 mL 0.2 M phosphate buffer (pH 6.6) usinga homogenizer. After centrifuging at 9070 gnfor 15 min, the supernatant was collected tomeasure the content of protein and aminoacid. Amino acid content was determinedusing the ninhydrin method (Yokoyama andHiramatsu, 2003). A 1% w/v ninhydrin stocksolution was prepared in ethanol containing0.025% w/v ascorbic acid. A working ninhy-drin solution was prepared immediately be-fore use by adding two parts of 0.4 M sodiumacetate buffer (pH 5.0) to one part of ninhy-drin stock solution. Extract or standard glu-tamate solution (50 mL) was added to 2.9 mLninhydrin work solution and the mixture washeated at 95 �C for 10 min. The solution wascooled and A570 was measured. Protein con-tent was determined according to the methodof Bradford (1976) using bovine serum albu-min as standard.

Statistical analysis. A complete random-ized design was used for this experiment.Each biological replicate contained one potand each treatment included four replicatepots for each trial. Treatment means wereseparated by Student’s t test at the 0.05 levelof probability using the JMP program version5 (SAS Institute Inc., Cary, NC). The in-teraction of the two trials was not significant,so data were pooled together.

Results

Soil physical and chemical properties.The granular vermicompost contained highlevels of macro- (N, P, K, Ca, Mg, and SO4)and micronutrients (Cu, Zn, Fe, Mn, and B),and organic matter and carbon (Table 1).However, it also had high levels of Na(9.8 g·kg–1) and Cl (13 g·kg–1) with high ECvalue (26 dS·m–1). The C:N ratio of solidvermicompost was 9.3. After harvesting, theamendments left high residual levels ofnutrients (P, K, SO4, Cu, Zn, Fe, and B),organic matter and carbon, and increased soilCEC and WHC. However, the soil EC in-creased from 0.58 to 0.92 and 1.4 dS·m–1 with5% and 10% amendments, respectively. Thedrench treatment increased, to a lesser extent,soil levels of P, Mg, SO4, Cu, Zn, Fe, and B.Drench treatment increased soil CEC andWHC, although it had negligible effects onorganic matter and carbon.

Growth and physiological responses.Compared with the control, all vermicomposttreatments (drench, 5Ver, and 10Ver) signif-icantly increased leaf number from 14.0 to15.3, 17.0, and 19.8 per plant (Fig. 1A); areafrom 155 to 252, 314, and 473 (Fig. 1B); FWfrom 8.7 to 14.3, 20.7, and 33 g per plant

Fig. 4. Effect of vermicompost on spinach specific leaf area (SLA; A), and succulence (B) 35 d aftertransplanting. The values are means of eight replicates ±SE. Different letters on top of bars indicatesignificant difference at P # 0.05 according to Student’s t test. Drench: 40 mL of vermicompostextract at 0, 14, 21, and 28 d after transplanting; 5Ver or 10Ver: soil amended with 5% or 10% (v/v)vermicompost.

Fig. 5. Effect of vermicompost on spinach chlorophyll (CHL) a (A), CHL b (B), and total CHL contents (C) 35 dafter transplanting. The values are means of eight replicates ±SE. Different letters on top of bars indicatesignificant difference at P# 0.05 according to Student’s t test. Drench: 40 mL of vermicompost extract at 0,14, 21, and 28 d after transplanting; 5Ver or 10Ver: soil amended with 5% or 10% (v/v) vermicompost.

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(Fig. 1C); and DW from 1.5 to 2.5, 3.3, and5.3 g per plant (Fig. 1D), respectively. ShootFW significantly increased from 10.6 to 17.5,24.4, and 39.6 g per plant (Fig. 2A); DW from1.8 to 3.1, 3.9, and 6.3 g per plant (Fig. 2C);and WUE from 3.4 to 5.8, 6.9, and11.3 mg·mL–1 H2O (Fig. 2B); by drench, 5Ver,and 10Ver treatments, respectively. ShootFW:DW ratio was unaffected by vermicom-post treatments (Fig. 2D).

Root DW significantly increased from0.56 to 0.84, 0.84, and 1.03 g per plant, inresponse to the drench, 5Ver, and 10Vertreatments, respectively (Fig. 3A). Shoot:root FW ratio increased from 3.3 to 4.9(5Ver) and 6.6 (10Ver), whereas the drenchtreatment had no effect (Fig. 3B). LeafRWC was not influenced by any vermicom-post treatments (data not shown). The 10Vertreatment decreased SLA, from 134 to 121cm2·g–1 DW (Fig. 4A). The two soil amend-ment treatments increased leaf succulence,from 413 to 505 and 510 H2O g·m–2, re-spectively (Fig. 4B).

Drench, 5Ver, and 10Ver significantlyincreased chlorophyll-a content from 2.7 to4.2, 3.5, and 3.6 mg·g–1 DW, respectively(Fig. 5A), chlorophyll b content from 0.83 to1.15, 1.22, and 1.16 mg·g–1 DW, respectively

(Fig. 5B), and total chlorophyll content from3.5 to 5.4, 4.7, and 4.8 mg·g–1 DW, respec-tively (Fig. 5C). All vermicompost treat-ments significantly increased Fv/Fm over thecontrol in the first, second, and third pairleaves, and increased Y(II) and ETR in thefirst and second pairs of leaves, but not in thethird pair of leaves (Fig. 6).

Nutritional values. Leaf carotenoid con-tent significantly increased from 1.4 to 1.9,1.7, and 1.8 mg·g–1 DW under drench, 5Ver,and 10Ver treatments, respectively (Fig. 7A).Soil amendments significantly decreased to-tal phenolic content from 17.6 to 14.9 and13.1 GAE mg·g–1 DW, respectively, whereasthe drench treatment had no effect (Fig. 7B).Betacyanin content significantly decreasedfrom 62 to 51, 38, and 32 mg·g–1 DW underdrench, 5Ver, and 10Ver treatments, respec-tively (Fig. 7C).

Soil amendments significantly reducedleaf flavonoid content from 3.3 to 2.8 (5Ver)and 2.5 (10Ver) CHE mg·g–1 DW (Fig. 8A),and total antioxidant capacity from 175 to153 and 147 TE mg·g–1 DW, respectively,whereas the drench treatment did not alterflavonoid content or antioxidant capacity(Fig. 8B). Compared with control, drenchand 10Ver treatment significantly increased

leaf amino acid content from 114 to 150 and158 mmol·g–1 DW, respectively (Fig. 9A).Protein content significantly increased from36 to 55, 47, and 59 mg·g–1 DW underdrench, 5Ver, and 10Ver treatments, re-spectively (Fig. 9B).

Discussion

Soil fertility. The vermicompost used inthe present study had preferable C:N ratioof 9.3. A C:N ratio less than 20 indicatesacceptable maturity of the product, buta ratio less than 15 is preferred (Gaur andSadasivam, 1993; Jimenez and Garcia,1992). Similar to previous studies (Forneset al., 2012; Padmavathiamma et al., 2008),the vermicompost has many favorableproperties including high content of or-ganic matter and carbon, and macro- andmicronutrients, in spite of the high ECvalue due to its high contents of Na andCl. This suggests that a high applicationrate as soil amendment is not recommendedbecause of the salinity stress it might cause.Even after harvesting, soil with vermicom-post amendments, especially at 10% rate,had high content of nutrients, organic matterand carbon, and high values of CEC andWHC. Also drench treatment increased soilnutrient contents, CEC, and WHC. Theresults indicate that vermicompost couldbe used to improve soil fertility as a soilamendment or drench.

Growth and physiological responses. Allvermicompost treatments, especially amend-ment at 10% rate, greatly stimulated spinachgrowth, as indicated by increased leaf number,area, FW and DW, shoot FW and DW, androot DW, and shoot growth was more favor-ably influenced than root growth by soilamendment. Peyvast et al. (2008a) reportedthat spinach plants with 10% vermicompost assoil amendment had highest leaf number, area,and FW. Numerous studies indicated thatvermicompost amendment into soilless mediain greenhouse resulted in increased germina-tion, growth and flowering of ornamentals,and growth and yield of vegetables even atlow mix rates (Arancon et al., 2008; Atiyehet al., 1999, 2000a, 2000b, 2001). In addition,Peyvast et al. (2008b) reported that vermicom-post as soil amendments enhanced parsley leafFW and DW, root DW, and plant height ingreenhouse. Similarly, in another greenhousestudy, vermicompost as soil amendments in-creased mustard root and shoot length, num-bers of branches, leaves, flowers and pods,and plant FW and DW (Srivastava et al.,2011). Favorable effects of vermicomposts assoil amendments have been reported in fieldstudies on the growth and yield of peppers,tomatoes, and strawberries (Arancon et al.,2003a, 2004, 2005a).

Although vermicompost contains macro-and micronutrients, its positive effects oncrop growth and yield may not be mainlydue to its nutrients, since in some studies,the nutrients in vermicompost were equal-ized in the control plots treated with in-organic fertilizers (Arancon et al., 2003a,

Fig. 6. Effect of vermicompost on spinach leaf photochemical efficiency (Fv/Fm; A), photochemical yield[Y(II); B], and electron transport rate (ETR; C) 35 d after transplanting. The values are means of eightreplicates ±SE. Different letters on top of bars indicate significant difference at P # 0.05 according toStudent’s t test. Drench: 40 mL of vermicompost extract at 0, 14, 21, and 28 d after transplanting; 5Veror 10Ver: soil amended with 5% or 10% (v/v) vermicompost.

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2004, 2005a; Atiyeh et al., 2000a, 2000c,2002a). The synergetic effects of manyfactors, such as improved physical structureof soil and nutrient availability, increasedmicrobial biomass and activities, suppressionof plant disease, insect and nematode, andbiologically active plant growth regulatorsand humic acid, may have been responsiblefor the growth stimulation by vermicompost.During vermicomposting process, microor-ganisms biochemically degrade organic mat-ter and earthworms physically fragment thesubstrate leading to new nutrient pools andlarge surface area, which provides nutrientsin forms that are readily taken up by theplants such as nitrates, exchangeable P, andsoluble K, Ca, and Mg (Orozco et al., 1996),and strong absorption capability and reten-tion of nutrients (Padmavathiamma et al.,2008; Srivastava et al., 2011). Vermicompostis a finely divided peat-like material, and itsaddition to soil can cause significant changes

in physical and chemical properties. Ourresults indicated that vermicompost en-hanced soil organic matter, CEC, and WHC.Similarly, Ferreras et al. (2006) reported thatvermicompost as soil amendment improvedsoil porosity and aggregate ability. Gopinathet al. (2008) observed decreases in soil bulkdensity and increases in soil pH and organiccarbon after addition of vermicompost. Thesechanges in soil properties improved the avail-ability of air and water, enhancing rootgrowth, which in turn facilitates water andnutrient absorption.

Earthworms can enhance microbial bio-mass and activities due to their mucus andcasts, which were responsible for betterlitter decomposition and mineralizationand provided high amount of availableforms of nutrients (Atiyeh et al., 2000b;Padmavathiamma et al., 2008; Srivastava et al.,2011; Tognetti et al., 2005). The byproductsof microbial activities, polysaccharides, can

help the aggregation of soil particles. Otherproducts of microbial activities includeplant growth–regulating substances such asauxins, gibberellins, cytokinins, ethylene,and abscisic acids (Arancon et al., 2012;Frankenberger and Arshad, 1995; Tomatiet al., 1987, 1988). In addition, humicmaterials from vermicomposts increasedplant growth of carrots, tomatoes, andpeppers (Arancon et al., 2003c, 2006b;Atiyeh et al., 2002b; Muscolo et al.,1999). Atiyeh et al. (2002b) and Aranconet al. (2003c, 2006b) suggested that humicacid may absorb plant hormones and/oritself has hormone ability to affect plantgrowth. Actually, Canellas et al. (2002)found that the identified auxin groups inhumic acids from vermicompost couldenhance root elongation, lateral root emer-gency, and plasma membrane H+-ATPaseactivity. Mora et al. (2010) observed thataction of humic acid on promotion of cucum-ber shoot growth involves nitrate-relatedchanges associated with the root-to-shootdistribution of cytokinins, polyamines, andmineral nutrients.

In addition, some studies have shown thatvermicompost can suppress a wide range ofmicrobial disease (Edwards et al., 2006),insect pest (Arancon et al., 2005b, 2007;Ramesh, 2000; Yardim et al., 2006), andparasitic nematodes (Arancon et al., 2003b;Swathi et al., 1998). As mentioned above,vermicompost increased soil microbial bio-mass and activity and changed the diversityand abundance of soil fauna, and thusa broader range of microorganisms may actas biocontrol agents through competition,antibiosis, and parasitism (Lazcano andDomínguez, 2011). Also, the induction ofplant systemic resistance by vermicompostwas observed by Singh et al. (2003).

All vermicompost treatments increasedspinach WUE, which might at least partlyresult from improved soil properties, such assoil WHC. Vermicompost as soil amend-ments reduced SLA at 10% mix rate andincreased leaf succulence, indicating theimprovement of spinach quality. There arelimited reports on chlorophyll content andphotochemistry of photosystem II as affectedby vermicompost. In this study, chlorophyllcontent increased under all vermicomposttreatments, especially drench treatment. Ver-micompost was also reported to increasechlorophyll content in marigold (Atiyehet al., 2001) and mustard (Srivastava et al.,2011) leaves. In the present study, vermicom-post treatments improved photochemistry ofphotosystem II, especially in mature leaves.They enhanced ETR and Y(II) of both firstand second pair leaves, and Fv/Fm of allleaves. Parameters related to photochemistryof photosystem II and chlorophyll content arecommonly used as indicators for leaf senes-cence (Adams III et al., 1990; Lima et al.,1999; Plesni�car et al., 1994). The resultsuggested that vermicompost could delayleaf senescence and extend leaf longevity.Similarly, the positive role of vermicompostin leaf production and longevity was reported

Fig. 7. Effect of vermicompost on spinach leaf carotenoid (A), total phenolic (PHE; B), and betacyanincontent (C) 35 d after transplanting. The values are means of eight replicates ±SE. Different letters ontop of bars indicate significant difference at P # 0.05 according to Student’s t test. Drench: 40 mL ofvermicompost extract at 0, 14, 21, and 28 d after transplanting; 5Ver or 10Ver: soil amended with 5%or 10% (v/v) vermicompost. GAE = gallic acid equivalents.

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in strawberry, banana, and cassava (Aranconet al., 2004; Padmavathiamma et al., 2008).High number of functional leaves is essentialfor crop production, especially for leafyvegetable such as spinach.

Nutritional values. Carotenoids have longbeen recognized as essential nutrients andimportant health-beneficial compounds(Fraser and Bramley, 2004). Consistent withprevious study in mustard and pak choi (Pantet al., 2009; Srivastava et al., 2011), itscontent increased under all vermicomposttreatments, especially under drench treat-ment, in the present study. However, vermi-compost as soil amendment, especially at10% rate, reduced the content of other phy-tochemicals such as phenolics, betacyanins,and flavonoids, and drench treatment onlyreduced betacyanin content in the presentstudy. Phenolics are a class of secondarymetabolites that play a key role as antioxi-dants. The most important group of pheno-lics in plants is flavonoids, which haveattracted considerable interest due to theirbroad spectrum of biological effects such asantioxidative, anti-inflammatory, vasorelax-ant, antimicrobial, antiviral, and for theiranticarcinogenic and antimutagenic activi-ties (Guo et al., 2011; Maimoona et al.,2011). In the present study, only vermicom-post as amendment decreased total antioxi-dant capacity, and the response pattern oftotal antioxidant capacity was very similarwith that of flavonoids, suggesting thatflavonoids might be the main antioxidantphytochemical in spinach leaves, at least ofthis cultivar. Its content might not be nega-tively altered by drench treatment of vermi-compost. There are a few reports aboutvermicompost’s effects on crop phytochem-ical content. Previous study indicated thatvermicompost tea decreased phenolic con-tent in pak choi (Pant et al., 2009). Zaller(2006) reported that ascorbic acid content intomato fruits decreased after foliar sprayingof vermicompost extract.

Vermicompost, whether as an amendmentor a drench, also favorably influenced spi-nach’s nutritional value in the present studyby increasing protein and amino acid con-tents. A previous study also indicated thatprotein content in mustard leaves was greatlyincreased by vermicompost (Srivastava et al.,2011). In addition, vermicompost was re-ported to increase sugar content in banana(Padmavathiamma et al., 2008), mustard(Srivastava et al., 2011), spinach, and parsley(Peyvast et al., 2008a, 2008b). Foliar appli-cation of vermicompost extract did not alterthe content of glucose or fructose in tomatofruit (Zaller, 2006). The information aboutvermicompost’s effect on mineral contentwas very limited and inconsistent, probablydue to different application methods. Ver-micompost leachate had no effects on N, P,and K concentration in sorghum leaves(Guti�errez-Miceli et al., 2008). Foliar appli-cation of vermicompost extract did not altermineral content of tomato fruit (Zaller,2006), whereas soil amendment increasedmineral content in spinach and parsley

Fig. 8. Effect of vermicompost on spinach leaf flavonoid content (FLA; A) and total antioxidant capacity (B)35 d after transplanting. The values aremeans of eight replicates ±SE. Different letters on top of bars indicatesignificant difference atP# 0.05 according to Student’s t test. Drench: 40mLof vermicompost extract at 0,14, 21, and 28 d after transplanting; 5Ver or 10Ver: soil amended with 5% or 10% (v/v) vermicompost.CHE = (+)-catechin hydrate equivalents; TE = trolox equivalents.

Fig. 9. Effect of vermicompost on spinach leaf amino acid (A) and protein content (B) 35 d after transplanting.The values aremeans of eight replicates ±SE. Different letters on top of bars indicate significant difference atP# 0.05 according to Student’s t test. Drench: 40 mL of vermicompost extract at 0, 14, 21, and 28 d aftertransplanting; 5Ver or 10Ver: soil amended with 5% or 10% (v/v) vermicompost.

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(Peyvast et al., 2008a, 2008b). The effect ofvermicompost on spinach sugar and mineralcontent was not assessed in the presentstudy, but deserves further investigation.

In summary, vermicompost improved soilfertility, prompted leaf production, delayedleaf senescence, enhanced spinach growthand WUE. It also improved spinach qualityby increasing succulence and the content ofcarotenoid, protein, and amino acid, althoughas soil amendment it reduced flavonoid con-tent, leading to low antioxidant capacity.Vermicompost as soil amendment or drenchfor spinach production in the field might bean efficient strategy for water savings, andorganic production, as well as recycling oforganic waste materials. Further researchshould be conducted to investigate its long-term effect and optimize its application ratesin the field.

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